1. Field of the Invention
The invention generally relates to the detection of homocysteine. In particular, the present invention provides rapid and sensitive methods for the detection and quantification of homocysteine in plasma and tissue.
2. Background of the Invention
Abnormally elevated levels of the amino acid homocysteine (hCys) in plasma and tissue extracts are related to many serious pathological conditions. For example, recent observations have demonstrated a relationship between hyperhomocysteinemia and coronary heart disease (1-3) and neural tube defects (4,5). Elevated levels of hCys are even more pronounced with other compromised organ or medical conditions, for example, heart transplants (6), end-stage renal disease (7), Parkinson's disease (8), diabetes (9), and preclampsia and fetal growth restriction (10). The exact relationship of elevated plasma hCys levels to these diseases is unknown and is the subject of intense investigation. The relationships are likely to be complex, as indicated by recent studies showing that hCys effects such diverse biological processes as mitochondrial gene expression (11), the secretion of cholesterol in hepatic cells (12), and leucovorin and 5-fluorouracil therapy in cancer patients (13).
Homocysteine is the precusor of the amino acid methionine and the metabolic pathways for the formation and breakdown of hCys are illustrated in FIG. 1. High levels of hCys can be caused by an impairment in any one of several enzymes involved in the metabolism of this amino acid. For example, a defect in the enzyme methionine synthase, which catalyzes Reaction 1 in FIG. 1, results in a buildup of hCys and inadequate supplies of methionine and adenosyl methionine (AdoMet). The inability to convert hCys to methionine can also be caused by an inadequate supply of either the substrate 5-methyltetrahydrofolate (5-CH.sub.3 -THF) or low vitamin B.sub.12, caused either by low folate nutrition or a defect in the enzyme methylenetetrahydrofolate reductase. Recent studies have demonstrated that low folate intake by pregnant women results in a several-fold increase in births of children with neural tube defects (4,5). The addition of folate to the diet frequently results in a lowering of the plasma hCys level. In some cases, both vitamin B.sub.12 and B.sub.6 can lower hCys levels. The effect of B.sub.6 is usually ascribed to the enzyme .beta.-cystathionase, an enzyme in the degradation pathway of hCys (Reaction 3 of FIG. 1).
As mentioned above, hyperhomocysteinemia is usually associated with low AdoMet levels. Low AdoMet levels can have devastating physiological consequences because AdoMet is the only methyl donor in the cell capable of methylating DNA, RNA, proteins, and numerous small molecules, many of which are precursors to neurotransmitters. (Although betaine can serve as a methyl donor in liver and kidney, this appears to be a minor pathway.) Thus, an individual with low AdoMet is at risk for a number of health problems.
Given the widespread health problems associated with elevated levels of hCys, it would be advantageous to have a rapid, inexpensive assay for detecting and quantifying hCys to be used in both clinical and research settings. The two major methods currently used do not fulfill these criteria in that they are both time-consuming and expensive.
Most of the hCys in plasma is protein-bound as disulfide and thus must be released by reduction prior to analysis. The most common currently-used method of detection reduces the hCys disulfide with dithiothreitol, enzymatically converts the hCys to S-adenosyl homocysteine, and detects the S-adenosyl homocysteine using monoclonal antibodies in an enzyme-immunosorbent assay (EIA)(14). The level of sensitivity of this assay is about 0.2 nmoles. The entire automated procedure, which has a 2.5 hour assay time, is presented in FIG. 2.
This method is available as a kit from Abbott Labs; the cost of each assay is about $10.00, not including the labor charge of the clinical lab making the measurement.
The second common procedure treats the reduced plasma with a derivatizing agent such as 4-aminosulfonyl-7-fluoro-2,1,3-benzoxydiazole, which forms a fluorescent adduct with the sulfhydryl group of hCys. (Note that dithiothreitol cannot be used in this assay because it interferes with subsequent HPLC analysis.) The fluorescent adduct of hCys is then separated by high performance liquid chromatography (HPLC) and the area under the peak corresponding to the hCys adduct is used to calculate the amount of hCys in the original plasma (15). This method requires 14 minutes to reduce the disulfide, 5 minutes for centrifugation, and up to 30 minutes for HPLC. Thus, nearly one hour is required to make a single determination. The level of sensitivity of this method is about 1 nmole. This procedure is available as a kit from BioRad at a cost of $7.00 to $7.50 per assay. For many labs doing research in this area, it is not uncommon to do 1000 hCys assays to obtain the data for a single publication. The cost would be at least $7000 using this method and require about 1000 hours of analysis time, since the procedure is not readily automated to accommodate large numbers of samples. Yet this is by far the method most often used in research laboratories.
The demand for assays of hCys is increasing. For example, the number of research publications about hCys has increased every year since 1990, from about 100 in 1990 to nearly 600 in 1998. During January and February of 1999 alone, over 100 hCys papers were published. This is clear evidence of a trend toward increased interest in hCys and the need for improved methods of assaying for hCys increases concomitantly. Likewise, hospital clinical labs such as that at the Medical College of Virginia anticipate that the demand for the analysis of hCys may approach that of cholesterol in the near future (private communication).
It would be a distinct advantage to have available rapid, sensitive and economically feasible methods for quantifying the level of hCys in biological samples. Such methods would be useful to both researchers in the scientific community and to clinical diagnosticians.